During infection by bacteriophage T4, the use of specific promoter sequences for the transcription of early, middle, and late genes correlates with different phage-induced modifications to the host polymerase. We are studying the expression of a cluster of T4 genes (5' -> 3'): x.1 (DNA endonuclease, related to intron-encoded-endonucleases), uvsX (recombination protein), 40 (Stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase) as a model for understanding how the phage regulates middle RNA. In vivo the T4 motA protein is required for transcription from T4 middle promoters. We have demonstrated that in vitro expression from the middle promoter located 190 bases upstream of uvsX is dependent on motA protein and middle polymerase (host RNA polymerase isolated from T4-infected cells at the start of middle gene expression, 4 min post-infection at 37 degrees) . RNA polymerase isolated from uninfected cells gives a minor amount of RNA from this promoter, but this synthesis is not motA-dependent. Previous studies have shown that middle polymerase differs from unmodified polymerase by the association of 2 phage proteins (10 kDa protein and 15 kDa, rbpA protein) and by the ADP-ribosylation of the alpha subunits. Thus, this in vitro transcription system provides the basis for a detailed study of the phage and host factors needed to regulate T4 middle gene expression. The biological function of the T4 X.1 gene is unknown since an T4 X.1 mutant is normal for phage growth and UV repair. We have found that the X.1 open reading frame shares tremendous similarity with 4 other T4 proteins of unknown function and that together these proteins share regions of homology with endonucleases of mobile group I introns. We have also found that, like these intron-encoded endonucleases, the X.1 protein is a Mg++-dependent DNA endonuclease with preferred sites for cutting. We Speculate that X.1 and the 4 other T4 ORFs represent a family of endonucleases related to the endonucleases present in mobile group I introns.